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Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR

Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR. Victoria A. Higman Leibniz-Institut für Molekulare Pharmakologie, Berlin and School of Chemistry, University of Bristol. O uter M embrane P rotein G. 281-residue membrane protein from E.Coli

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Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR

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  1. Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR Victoria A. Higman Leibniz-Institut für Molekulare Pharmakologie, Berlin and School of Chemistry, University of Bristol

  2. Outer Membrane Protein G • 281-residue membrane protein from E.Coli • b-barrel fold, monomeric porin • expressed in the absence of genes for other porins • channel for mono-, di- and tri-saccharides • possibly pH-gated via extracellular loop  of biotechnological interest Membrane Membrane

  3. refolding into detergent containing buffer concentration and addition of E.Coli lipid extract 2D crystallisation by dialysis Ultracentrifugation Sample Preparation expression in inclusion bodies

  4. Cb N C Ca N C C Ca N C Ca Cb Cb 13C-13C Correlation

  5. Cb N C Ca N C C Ca N C Ca Cb Cb 13C-13C Correlation 1 – all carbons (CP) CP – cross polarisation

  6. Cb C N C Ca N C C Ca N C Ca C Cb Cb 13C-13C Correlation 1 – all carbons (CP) 2 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  7. 13C-13C Correlation Ala Ca Ala Cb

  8. Cb N C Ca N C C Ca N C Ca Cb Cb 13C-13C Correlation 20 ms C C 200-700 ms 1 – all carbons (CP) 2 – other carbons (PDSD)

  9. 13C-13C Correlation GAFY-OmpG PDSD 25ms DARR700ms 900 MHz 13 kHz MAS

  10. Cb N C Ca N C C Ca N C Ca Cb Cb NCACX

  11. Cb N C Ca N C C Ca N C Ca Cb Cb NCACX 1 – Nitrogen (CP) CP – cross polarisation PDSD – proton driven spin diffusion

  12. Cb N C Ca N C C Ca N C Ca Cb Cb NCACX 1 – Nitrogen (CP) 2 – Ca (CP) CP – cross polarisation PDSD – proton driven spin diffusion

  13. Cb Ca N N C Ca N C C Ca N C Ca other carbons Cb Cb NCACX 1 – Nitrogen (CP) 2 – Ca (CP) 3 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  14. Ca N other carbons NCACX

  15. Cb Ca N N C Ca N C C Ca N C Ca other carbons Cb Cb NCACX 20 ms 200-500 ms 1 – Nitrogen (CP) 2 – Ca (CP) 3 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  16. Cb N C Ca N C C Ca N C Ca Cb Cb NCOCX

  17. Cb N C Ca N C C Ca N C Ca Cb Cb NCOCX 1 – Nitrogen (CP) CP – cross polarisation

  18. Cb N C Ca N C C Ca N C Ca Cb Cb NCOCX 1 – Nitrogen (CP) 2 – CO (CP) CP – cross polarisation

  19. Cb CO N N C Ca N C C Ca N C Ca other carbons Cb Cb NCOCX 1 – Nitrogen (CP) 2 – CO (CP) 3 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  20. Cb CO N N C Ca N C C Ca N C Ca other carbons Cb Cb NCOCX 1 – Nitrogen (CP) 2 – CO (CP) 3 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  21. Cb Ca N N C Ca N C C Ca N C Ca Cb Cb Cb NCACB 1 – Nitrogen (CP) 2 – Ca (CP) 3 – Cb (DREAM) CP – cross polarisation DREAM – dipolar recoupling enhancement through amplitude modulation

  22. Cb Ca N N C Ca N C C Ca N C Ca CO Cb Cb CANCO 1 – Ca (CP) 2 – N (CP) 3 – CO (CP) CP – cross polarisation

  23. Cb N C Ca N C C Ca N C Ca Cb Cb CANCOCX CO Ca N other carbons 1 – Ca (CP) 2 – N (CP) 3 – CO (CP) 4 – other carbons (PDSD) CP – cross polarisation PDSD – proton driven spin diffusion

  24. Assignment Strategy • Identification of Spin Systems 13C-13C Correlation NCACX NCACB • Linking of Spin Systems 13C-13C Correlation NCACX NCOCX CANCO / CANCOCX

  25. OmpG - Initial Analysis U-[13C-15N] OmpG PDSD (25ms) 900 MHz 13 kHz MAS

  26. 13C OH C OH OH 13C [1,3-13C]- glycerol C OH 13C OH C OH [2-13C]- glycerol Labelling with Glycerol

  27. Labelling with Glycerol Glycerol His Ser Gly Phe, Tyr, Trp Cys Val, Leu Ala Asn Asp Glu Pro Gln Arg Lys Met Thr Ile

  28. Labelling with Glycerol • Sharper signals • More long-range cross peaks • Less overlap

  29. Labelling with Glycerol U-[13C] OmpG PDSD (25ms) [1,3-13C]-glycerol OmpG PDSD (50ms) 900 MHz, 13 kHz MAS

  30. Labelling with Glycerol U-[13C] OmpG PDSD (25ms) [1,3-13C]-glycerol OmpG PDSD (50ms) 900 MHz, 13 kHz MAS

  31. Labelling with Glycerol U-[13C] OmpG PDSD (25ms) [2-13C]-glycerol OmpG PDSD (50ms) 900 MHz, 13 kHz MAS

  32. Identifying Spin Systems [2-13C]-glycerol OmpG, PDSD (50ms) 900 MHz, 13kHz MAS

  33. Identifying Spin Systems [2-13C]-glycerol OmpG, PDSD (50ms) 900 MHz, 13kHz MAS

  34. Identifying Spin Systems [2-13C]-glycerol OmpG, PDSD (50ms) 900 MHz, 13kHz MAS

  35. Identifying Spin Systems [2-13C]-glycerol OmpG, PDSD (125ms) 900 MHz, 13kHz MAS

  36. Identifying Spin Systems

  37. Identifying Spin Systems

  38. Identifying Spin Systems

  39. Identifying Spin Systems

  40. Linking Spin Systems

  41. Linking Spin Systems

  42. Linking Spin Systems [1,3-13C]-labelled NCACX / NCOCX

  43. Linking Spin Systems [1,3-13C]-labelled NCACX / NCOCX [2-13C]-labelled NCACX / NCOCX

  44. Linking Spin Systems [1,3-13C]-labelled NCACX / NCOCX [2-13C]-labelled NCACX / NCOCX

  45. Linking Spin Systems [1,3-13C]-labelled NCACX / NCOCX [2-13C]-labelled NCACX / NCOCX

  46. LeuCO ThrN ThrCa LeuCb LeuCg Cd Cd Cg Cb Ca N C N C Ca Cb Thr Leu Cg Assignment [2-13C]-glycerol OmpG, NCOCX (50ms), 400 MHz, 8 kHz MAS

  47. Assignment – SH3

  48. Assignment – aB-crystallin

  49. Labelling with Glycerol

  50. Glucose His Ser Gly Phe, Tyr, Trp Cys Val, Leu Ala Asn Asp Glu Pro Gln N C N N N C C C Lys Arg Met Ca Ca Ca Ca Thr Cb Cb Cb Ile [2,3-13C] Labelling – GAFY GAFY Phe Tyr Gly Ala

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